Major neuropsychiatric disorders, including Aids Dementia, Alzheimer's Disease, depression and schizophrenia, may involve aberrant phagocytosis of cerebral cortical synapses by monocytes and/or microglia. However, efforts to target this process for therapeutic intervention have been limited. One reason for the limited success in the development of therapeutic interventions for CNS microglia/macrophage activation is that species differences between the immune systems of humans and model organisms render the co-culture of human immune cells with non-human neurons less informative. Fortunately, recent progress in generating cortical cells, including neurons, astrocytes and microglia, from human stem cells provides a new opportunity to address this challenge. This proposal is a collaborative effort between the labs of Kelly Jordan-Sciutto, who studies neuro-AIDS and monocyte activity in vitro, and Stewart Anderson, who studies forebrain development in relation to neuropsychiatric disorders using mouse and human stem cells. Differentiation of iPSCs into forebrain-like excitatory neurons (iNrns), and their co-culture with astrocyte-like cells (iAstrs), results in a synaptically dense network. By plating human monocytes over those neurons we observe IBA1+ cells that appear to have ingested synaptic markers. Moreover, we are able to generate human microglia like cells (iMgl), infect them with HIV, and find that they react to this infection by cytokine release and the formation of multinucleated cells. Importantly, exposure to an antiretroviral agent reduces their reverse transcriptase activity. We also find that we can co-culture iMgl together with i-Nrns and i-Astrs. In this exploratory R21 we will fine-tune this human iMgl, iAstr, iNrn culture system to test the hypothesis that HIV infected microglia decrease excitatory synapse density in vitro. If so, we will determine whether the effect is mimicked by conditioned media, is associated with increased compliment deposition, and is affected by ART. We will also test whether viral suppression occurs in the iMgl within the triculture context. Success would lead to new approaches for mechanistic studies and therapeutics development for the multiple neuropsychiatric disorders, such as HIV Associated Dementia, in which monocyte/microglial activation may play a crucial role in their pathogenesis.